Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
This technology has progressed extremely rapidly in recent years and a variety of exogenous proteins have been expressed in a variety of hosts, but obtaining any desired novel cDNA clone remains an uncertainty. By way of example, some of the eukaryotic proteins produced by recombinant DNA technology include: proinsulin (Naber, S. et al., Gene 21:95-104 [1983]); interferons (Simon, L. et al., Proc. Natl. Acad. Sci. U.S.A., 80:2059-2062 [1983] and Derynck, R. et al., Nucl. Acids Res. 1:1819-1837 [1983]); growth hormone (Goeddel, D., et al., Nature 281:544-548 [1979]); a human T-cell growth factor (Taniguichi, T. et al., Nature 302:305-310 (1983)); and a granulocyte/macrophage cellular growth factor (G/M-CSF) (Miyatake, S. et al., EMBO J. 4:2561-2568 (1985)). These publications and other reference materials cited hereafter have been included to provide additional details on the background of the pertinent art and, in particular instances, the practice of the invention, and are all incorporated herein by reference.)
For some time, it has been documented that the mammalian immune response is based on a series of complex cellular interactions, called the "immune network." Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the response does, in fact, revolve around the network-like interactions of lymphocytes, macrophages, granulocytes and other cells, immunologists now generally hold the opinion that soluble proteins (e.g., the so-called "lymphokines") play a critical role in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which should yield significant breakthroughs in the diagnosis and therapy of numerous disease states.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth and the differentiation of the pluripotential hematopoietic stem cells into the vast number of progenitors composing the diverse cellular lineages responsible for the immune response. These lineages often respond in a different manner when lymphokines are used in conjunction with other agents.
Cell lineages important to the immune response include two classes of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal), and T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and some of the other cells (including other T-cells) making up the immune network.
Another important cell lineage is the mast cell (which has not been positively identified in all mammalian species)--a granule-containing connective tissue cell located proximal to capillaries throughout the body, with especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows: when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases the mediators (e.g., histamine, serotonin, heparin, kinans, prostaglandins, etc.) which cause allergic reactions, e.g., anaphylaxis, as well as others.
Research to better understand (and thus potentially treat therapeutically) various immune disorders has been hampered by the general inability to maintain in vitro cells of the immune system. Immunologists have discovered that culturing these cells can be accomplished through the use of T-cell and other cell supernatants, which contain various growth factors, such as some of the lymphokines.
The detection, isolation and purification of these factors is extremely difficult, being frequently complicated by the complexity of the supernatants they are typically located in, the divergencies and cross-overs of activities of the various components in the mixtures, the sensitivity (or lack thereof) of the assays utilized to ascertain the factors' properties, and the frequent similarity in the range of molecular weights and other characteristics of the factors.
Clarification of these issues requires additional structural data, e.g., substantially full-length sequence analysis of the molecules in question. Protein sequencing offers, of course, a possible means to solve the problem, but it is very difficult work experimentally and often can provide neither completely accurate nor full-length amino acid sequences. Moreover, having the capability of making bulk quantities of a polypeptide exhibiting activity on the cells of the immune system will greatly facilitate the study of the biology of those and other cells, e.g., by minimizing the necessity of relying on lectin conditioned media for stimulating cell growth. Accurate and complete sequence data on any immune protein will also serve to simplify the search for other immunological factors. Finally, additional information on any lymphokine will help in evaluating the roles of the various growth factors and cells of the immune network and thus provide insight into the entire immune system--with the concomitant therapeutic benefits.
Thus, there exists a significant need for extensive nucleotide sequence data on the DNAs coding for, and amino acid sequences of, proteins exhibiting immune cell growth or stimulatory activity, as well as a simple and economic method of making substantial and essentially pure quantities of such materials. The present invention fulfills these needs.